1. Field of the Invention
The present invention relates generally to the treatment of cancer. More particularly, it concerns novel compounds useful for chemotherapy, methods of synthesis of these compounds and methods of treatment employing these compounds. The novel compounds are halogenated anthracyclines related to anthracyclines such as daunorubicin and doxorubicin which are known to have antitumor activity. The use of these compounds in the treatment of Alzheimer""s disease are also contemplated.
2. Description of Related Art
Resistance of tumor cells to the killing effects of chemotherapy is one of the central problems in the management of cancer. It is now apparent that at diagnosis many human tumors already contain cancer cells that are resistant to standard chemotherapeutic agents. Spontaneous mutation toward drug resistance is estimated to occur in one of selective pressure from drug therapy, although radiation therapy and chemotherapy may give rise to additional mutations and contribute to tumor progression within cancer cell populations (Goldie et al., 1979; Goldie et al., 1984; Nowell, 1986). The cancer cell burden at diagnosis is therefore of paramount importance because even tumors as small as 1 cm (109 cells) could contain as many as 100 to 1,000 drug-resistant cells prior to the start of therapy.
Selective killing of only the tumor cells sensitive to the drugs leads to an overgrowth of tumor cells that are resistant to the chemotherapy. Mechanisms of drug resistance include decreased drug accumulation (particularly in multi-drug resistance), accelerated metabolism of the drug and other alterations of drug metabolism, and an increase in the ability of the cell to repair drug-induced damage (Curt et al., 1984; and Kolate, 1986). The cells that overgrow the tumor population not only are resistant to the agents used but also tend to be resistant to other drugs, many of which have dissimilar mechanisms of action. This phenomenon, called pleiotropic drug resistance or multi-drug resistance (MDR), may account for much of the drug resistance that occurs in previously treated cancer patients. The development of drug resistance is one of the major obstacles in the management of cancer. One of the traditional ways to attempt to circumvent this problem of drug resistance has been combination chemotherapy.
Combination drug therapy is the basis for most chemotherapy employed to treat breast, lung, and ovarian cancers as well as Hodgkin""s disease, non-Hodgkin""s lymphomas, acute leukemias, and carcinoma of the testes. Combination chemotherapy uses the differing mechanisms of action and cytotoxic potentials of multiple drugs.
Although combination chemotherapy has been successful in many cases, the need still exists for new anti-cancer drugs. These new drugs could be such that they are useful in conjunction with standard combination chemotherapy, or these new drugs could attack drug, resistant tumors by having the ability to kill cells of multiple resistance phenotypes.
A drug that exhibits the ability to overcome multiple drug resistance could be employed as a chemotherapeutic agent either alone or in combination with other drugs. The potential advantages of using such a drug in combination with chemotherapy would be the need to employ fewer toxic compounds in the combination, cost savings, and a synergistic effect leading to a treatment regime involving fewer treatments.
The commonly used chemotherapeutic agents are classified by their mode of action, origin, or structure, although some drugs do not fit clearly into any single group. The categories include alkylating agents, anti-metabolites, antibiotics, alkaloids, and miscellaneous agents (including hormones). Agents in the different categories have different sites of action.
Antibiotics are biologic products of bacteria or fungi. They do not share a single mechanism of action. The anthracyclines daunorubicin and doxorubicin (DOX) are some of the more commonly used chemotherapeutic antibiotics. The anthracyclines achieve their cytotoxic effect by several mechanisms, including inhibition of topoisomerase II; intercalation between DNA strands, thereby interfering with DNA and RNA synthesis; production of free radicals that react with and damage intracellular proteins and nucleic acids; chelation of divalent cations; and reaction with cell membranes. The wide range of potential sites of action may account for the broad efficacy as well as the toxicity of the anthracyclines (Young et al., 1985).
The anthracycline antibiotics are produced by the fungus Streplonlyces peuceitius var. caesius. Although they differ only slightly in chemical structure, daunorubicin has been used primarily in the acute leukemias, whereas doxorubicin displays broader activity against human neoplasms, including a variety of solid tumors. The clinical value of both agents is limited by an unusual cardiomyopathy, the occurrence of which is related to the total dose of the drug; it is often irreversible. In a search for agents with high antitumor activity but reduced cardiac toxicity, anthracycline derivatives and related compounds have been prepared. Several of these have shown promise in the early stages of clinical study, and some, like epirubicin and idarubicin, are used as drugs. Epirubicin outsells doxorubucin in Europe and Japan, but it is not sold in the U.S.
The anthracycline antibiotics have tetracycline ring structures with an unusual sugar, daunosamine, attached by glycosidic linkage. Cytotoxic agents of this class all have quinone and hydroquinone moieties on adjacent rings that permit them to function as electron-accepting and donating agents. Although there are marked differences in the clinical use of daunorubicin and doxorubicin, their chemical structures differ only by a single hydroxyl group on C14. The chemical structures of daunorubicin and doxorubicin are shown in FIG. 1.
Doxorubicin""s broad spectrum of activity against most hematological malignancies as well as carcinomas of the lung, breast, and ovary has made it a leading agent in the treatment of neoplastic disease (Arcamone, 1981; Lown, 1988; Priebe, 1995). Since the discovery of daunorubicin and doxorubicin (FIG. 1), the mechanistic details of the antitumor activity of anthracycline antibiotics have been actively investigated (Priebe, 1995; Priebe, 1995; Booser, 1994).
Studies have shown that the anthracycline, 4xe2x80x2-iodo-4xe2x80x2-deoxydoxorubicin (IDOX), binds strongly to amyloid fibrils. Preincubation of the amyloid enhancing factor with IDOX significantly reduces formation of amyloid deposits (Merlini et al., 1995). Amyloid fibril formation is involved in a number of diseases, including amyloidosis, prion diseases and Alzheimer""s disease. Amyloidosis is a rapidly progressive disease, characterized by the tissue deposition of paraproteins as insoluble fibrils, leading to organ dysfunction and death. Patients with amyloidosis, showed substantial clinical improvement, as a result of amyloid resorption, when treated with IDOX (Gianni et al., 1995). Prion diseases are characterized by the accumulation of protease-resistant insoluble forms of the prion-protein into aggregates of amyloid fibrils in the brain. In tests on an experimental Syrian-hamster model of prion disease, IDOX treated hamsters had a delayed onset of the disease and their survival time was prolonged. Neuropathologial examination of the treated hamster brains showed a parallel delay in the accumulation of amyloid fibrils with respect to the untreated controls (Tagliavini et al., 1997).
Unfortunately, concomitant with its antitumor and anti-amyloidogenic activities, DOX can produce adverse systemic effects, including acute myelosuppression, cumulative cardiotoxicity, and gastrointestinal toxicity (Young et al., 1985). At the cellular level, in both cultured mammalian cells and primary tumor cells, DOX can select for multiple mechanisms of drug resistance that decrease its chemotherapeutic efficacy. These mechanisms include P-gp-mediated MDR, characterized by the energy-dependent transport of drugs from the cell (Bradley et al., 1988), and resistance conferred by decreased topoisomerase II activity, resulting in the decreased anthracycline-induced DNA strand scission (Danks et al., 1987; Pommier et al., 1986; Moscow et al., 1988.
Among the potential avenues of circumvention of systemic toxicity and cellular drug resistance of the natural anthracyclines is the development of semi-synthetic anthracycline analogues which demonstrate greater tumor-specific toxicity and less susceptibility to various forms of resistance.
The present invention seeks to overcome drawbacks inherent in the prior art by providing compositions of agents that display increased cytotoxicity when compared with doxorubicin and can prevent and/or overcome multi-drug resistance. This invention involves novel compounds that have utility as antitumor and/or chemotherapeutic drugs, methods of synthesizing these compounds and methods of using these compounds to treat patients with cancer. The invention is generally based on the discovery that anthracycline derivatives that have fluorine groups attached to their sugar moiety have a surprisingly strong ability to kill tumor cells.
The inventors have designed halogenated anthracyclines, as exemplified by fluorinated anthracyclines, connected at positions which would not interfere with DNA binding, and the inventors have synthesized anthracyclines having sugar portions that have been halogenated at positions 4xe2x80x2 and 6xe2x80x2. These actions produced halogenated anthracyclines which exhibit activity substantially different from the activities of doxorubicin or daunorubicin. These compounds are active against doxorubicin resistant tumors and/or are more cytotoxic than doxorubicin against sensitive tumors. This indicates that halogenated anthracyclines are mechanistically different from doxorubicin and daunorubicin.
In some specific embodiments, the anthracycline compounds of the present invention have the general formula: 
wherein: R1 is a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3), an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)nCH3, wherein n=an integer from 1 to about 20, or a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3, wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9; each of R2 and R3 is, independently of the other, a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3) or a double bonded oxygen moiety; R4 is a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3) or a halide; each of Y1 and y2 is, independently of the other, a hydrogen (xe2x80x94H) group; a hydroxyl group (xe2x80x94OH); a methoxy group (xe2x80x94OCH3); or a double bonded oxygen, sulphur, or nitrogen group; R5 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, or xe2x80x94NHR11; R6 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NR112, xe2x80x94NR112, or xe2x80x94NHR11; R7 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, xe2x80x94NHR11, F, I, Br, or Cl, with the proviso that R7 can be I only when R6 is xe2x80x94OH or xe2x80x94SH; R8 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, xe2x80x94NHR11, F, I, Br, or Cl; R9 is CH3, CH2F, CH2I, CH2Br, or CH2Cl; R10 is H, F, I, Br, or Cl; and R11 is a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3), an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)nCH3, wherein n=an integer from 1 to about 20, or a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3, wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9.
Certain specific embodiments of the anthracyclines of the invention are: 
Specific embodiments of the anthracylines of the invention may have the structures shown in FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 9, FIG. 11, FIG. 14, or FIG. 19.
The present application also comprises methods of preparing anthracyclines. In general, the methods comprise a step of preparing or obtaining an anthracycline of formula: 
wherein: R1 is a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3), an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)nCH3, wherein n=an integer from 1 to about 20, or a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3, wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9; each of R2 and R3 is, independently of the other, a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3) or a double bonded oxygen moiety; R4 is a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3) or a halide; each of Y1 and y2 is, independently of the other, a hydrogen (xe2x80x94H) group; a hydroxyl group (xe2x80x94OH); a methoxy group (xe2x80x94OCH3); or a double bonded oxygen, sulphur, or nitrogen group. The methods further comprise a step of obtaining or preparing a saccharide of general formula: 
wherein: R5 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, or xe2x80x94NHR11 or N3; R6 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, NR112, or xe2x80x94NHR11 or N3; R7 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, xe2x80x94NHR11, N3, F, I, Br, or Cl, with the proviso that R7 can be I only when R6 is xe2x80x94OH or xe2x80x94SH;R8 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, xe2x80x94NHR11, N3, F, I, Br, or Cl; R9 is CH3 CH2F, CH2I, CH2Br, or CH2Cl; R10 is H, F, I, Br, or Cl; R11 is a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3), an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)nCH3, wherein n=an integer from 1 to about 20, or a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3, wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9; and R12 is an alkyl group, an S-alkyl group, an acyl group, an S-acyl group, a silylalkyl group, a halide, or any other leaving group. The anthracycline is then conjugated to the saccharide under suitable conditions to produce a glycosidic bond and result in a compound of the formula: 
In some preferred embodiments, the method of anthracycline synthesis comprises steps of synthesizing a 4 or 6 halogen-substituted sugar moiety; conjugating said sugar moiety to a doxorubicin or daunorubicin analog via a glycosidic bond; removing extraneous solvent to obtain a crude halogenated anthracycline; and purifying the crude halogenated anthracycline.
The invention encompasses novel methods of chemical synthesis wherein a glycosyl donor saccharide having a 3xe2x80x2 or 4xe2x80x2 amine group masked as an azide is employed. In a specific example of such a synthesis, one may couple an anthracycline to a saccharide by obtaining an anthracycline, obtaining a saccharide which has a 3xe2x80x2 or 4xe2x80x2 amine group masked as an azide group, conjugating the anthracycline to the saccharide via a glycosidic bond, and reducing the azide to an amine to obtain a 3xe2x80x2- or 4xe2x80x2-amine anthracycline. In some preferred embodiments, the amine group is placed at the 3xe2x80x2 position on the saccharide. Some preferred saccharides for the practice of this method are halogenated sugars comprising the structure: 
wherein: R1 is any suitable leaving group that will allow for formation of a glycosidic bond; R2, R3, and R4 are: H, OH, OR5, SH, SR5, NHR5, NH2, NR52, F, I, Br, Cl, and R5 is: a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3), an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)nCH3, wherein n=an integer from 1 to about 20, or a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3, wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9.
In a more general aspect, the invention comprises glycosyl donors having a structure: 
wherein: R5 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, or xe2x80x94NHR11; R6 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, NR112, or xe2x80x94NHR11; R7 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, xe2x80x94NHR11, F, I, Br, or Cl, with the proviso that R7 can be I only when R6 is xe2x80x94OH or xe2x80x94SH; R8 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, xe2x80x94NHR11, F, I, Br, or Cl; R9 is CH3 CH2F, CH2I, CH2Br, or CH2Cl; R10 is 14, F, I, Br, or Cl; R11 is a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3), an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)nCH3, wherein n=an integer from 1 to about 20, or a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)n(CH2)nCH3, wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9; and R12 is an alkyl group, an S-alkyl group, an acyl group, an S-acyl group, a silylalkyl group, a halide, or any other leaving group.
The above-described method of forming a compound comprising a glycosyl donor conjugated to an aglycon through a glycosyl bond comprising obtaining a glycosyl donor having a structure: 
wherein: R5 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, or xe2x80x94NHR11; R6 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, NR112, or xe2x80x94NHR11; R7 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, xe2x80x94NHR11, F, I, Br, or Cl, with the proviso that R7 can be I only when R6 is xe2x80x94OH or xe2x80x94SH; R8 is xe2x80x94H, xe2x80x94OH, xe2x80x94OR11, xe2x80x94SH, xe2x80x94SR11, xe2x80x94NH2, xe2x80x94NHR11, F, I, Br, or Cl; R9 is CH3, CH2F, CH2I, CH2Br, or CH2Cl; R10 is H, F, I, Br, or Cl; R11 is a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3), an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)nCH3, wherein n=an integer from 1 to about 20, or a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3, wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9; and R12 is an alkyl group, an S-alkyl group, an acyl group, an S-acyl group, a silylalkyl group, a halide, or any other leaving group; obtaining an aglycon; and forming a glycosidic bond between the glycosyl donor and the aglycon. The aglycon can be any form of biological molecule.
The present invention also comprises halogenated sugars that are useful for a variety of drug and chemical synthesis purposes. Preferably, these sugars are halogenated at the 4xe2x80x2 or the 6xe2x80x2 position. Generally, exemplary halogenated sugars may comprise the structure: 
wherein: R1 is any suitable leaving group that will allow for formation of a glycosidic bond; R2, R3, and R4 are: H, OH, OR5, SH, SR5, NHR5, NH2, NR52, F, I, Br, Cl, or a sugar; and R5 is: a hydrogen (xe2x80x94H) group, a hydroxyl group (xe2x80x94OH), a methoxy group (xe2x80x94OCH3), an aryl group having 1-20 carbon atoms, a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)nCH3, wherein n=an integer from 1 to about 20, or a fatty acyl group having the general structure xe2x80x94Oxe2x80x94CO(CH2)l(CHxe2x95x90CH)m(CH2)nCH3, wherein l is an integer between 1 to 3, m is an integer between 1 and about 6, and n is an integer between 1 to about 9.
Several specific exemplary halogenated sugars are shown in FIG. 17.
The invention also relates to methods of obtaining derivatives of DOX by the use of azides as blocking groups and linking an anthracycline to a saccharide. The steps required include obtaining an anthracycline; obtaining a saccharide which is masked by an azide group; conjugating the anthracycline to the saccharide via a glycosidic bond; and reducing the azide to an amine to obtain an amine anthracycline.
Another important embodiment of this invention is a method for treating Alzheimer""s disease comprising obtaining an anthracycline compound as described above, and administering to a person that either has Alzheimer""s disease or has the predisposition for Alzheimer""s disease, a pharmaceutically acceptable formulation of the anthracycline compound in a dose effective for the treatment of Alzheimer""s disease. The treatment results in curing, improving, or preventing Alzheimer""s disease in a person. In another aspect the invention comprises obtaining the compound described above and administering the compound, in addition to another anti-Alzheimer""s drug, to a person having or at the risk of developing Alzheimer""s disease in an amount effective to cure, improve, and/or prevent Alzheimer""s disease.
In devising the synthetic schemes and compounds of the present invention, the inventors have created a variety of novel compounds. These compounds are described elsewhere in the specification and figures, and are given xe2x80x9cWPxe2x80x9d numbers. The structure of a compound designated with a xe2x80x9cWPxe2x80x9d number is ascertainable by reviewing the specification and figures. Exemplary specific compounds that are encompassed by the invention are WP351, WP556, WP557, WP559, WP564, WP563, WP715, WP722, WP745, WP587, WP588, WP589, WP590, WP592, WP600, WP610, WP743, WP458, WP508, and WP526. Also encompassed by the invention are 6xe2x80x2-F-epidaunorubicin and 6xe2x80x2-F-epirubicin.
The invention also considers methods of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of the contemplated halogenated anthracycline compounds and therapeutic kits comprising, in suitable container means, a pharmaceutically acceptable composition comprising the contemplated halogenated anthracycline compounds.